Myoglobin and

hemoglobin
Lecture 3
Dr. Malik ALQUB MD. PHD.
 Hemoglobin and Myoglobin
•First protein to be crystallized - 1849.
•First protein to have its mass accurately
measured.
•First protein to be studied by
ultracentrifugation.
•First protein to be associated with function
•First protein to show that a point mutation can
cause a disease.
•First proteins to have his X-ray structure.
Structure of myoglobin
 The heme group

Fe in Mb is Fe2+ - ferrous iron - the

form that binds oxygen

+
Protoporphyrin IX
Overveiw of heme synthesis
Heme

Succinyl CoA + Glycine Protoporphyrin IX

ALA synthase
Protoporphyrinogen IX
-aminolevulinic acid

Coproporphyrinogen III

mitochondrial matrix cytoplasm

-aminolevulinic acid

Porphobilinogen Uroporphyrinogen III Coproporphyrinogen III

Uroporphyrinogen I Coproporphyrinogen I

Heme synthesis occurs in all cells due to the requirement for heme as a prosthetic group on
enzymes and electron transport chain. By weight, the major locations of heme synthesis are
the liver and the erythroid progenitor cells of the bone marrow.
Binding of oxygen induce
conformational changes
Binding of oxygen induce conformational
changes
 The Conformation Change

Oxygen binding changes the Mb conformation

Without oxygen bound, Fe is out of heme plane
Oxygen binding pulls the Fe into the heme plane
Fe pulls its His F8 ligand along with it
The F helix moves when oxygen binds
Total movement of Fe is 0.029 nm - 0.29 A
This change means little to Mb, but lots to Hb!
Hemoglobin structure
 How is oxygen carried ?

Complex protein containing heme (iron)

1 molecule of hemoglobuline combines with 4 molecules
of oxygen to form oxyhaemoglobin

Hb + O2 HbO8
Haemoglobin oxygen oxyhaemoglobin

This reaction is reversible.

Cooperativity
The haemoglobin molecule is a tetramer composed
of 4 protein subunits, each of which can bind a
single molecule to O2
These subunits exhibit cooperativity when they
bind to O2
i.e. the binding of O2 to any subunits increases the
likelihood that the other subunits will bind to O2
Hemoglobin structure
 Oxygen-Hemoglobin
Dissociation Curve

Shows the amount of

O2 that is bound to
haemoglobin (Y-axis)
as a function of the
partial pressure of O2 in
the blood plasma (X-
axis).
Because of the
cooperativity, this
dissociation curve is S-
shaped
 Significance of the S-shape curve
100%

Plateau:
► haemoglobin highly saturated
% saturation of ► favour the loading of O2 in lung
haemoglobin

Steep slope:
► small drop of O2 partial pressure leads to a rapid
decrease in % saturation of haemoglobin
► favour the release of O2 in tissue cells
partial pressure
of O2 (mmHg)
Body Lung
tissue
 Shift to right= low affinity to oxygen
100%

% saturation of
haemoglobin
50%

partial pressure
of O2 (mmHg)
26
mmHg
 Shift to left= high affinity to oxygen
100%

% saturation of
haemoglobin
50%

partial pressure
of O2 (mmHg)
26
mmHg
Hemoglobin structure
Binding of CO2 to hemoglobin

•Increase the stability of hemoglobin (T form)

•Decrease the affinity of hemoglobin to oxygen

•Shift the Dissociation Curve to right

Binding of CO to hemoglobin

•Increase the (R form)

•Increase the affinity of

hemoglobin to oxygen
PH and Oxygen-Hemoglobin
Dissociation Curve (bohr effect)
About 70% of the blood

CO2 reacts with H2O to form carbonic acid

CO2 + H2O  H2CO3

Carbonic acid rapidly dissociates into

Carbonic
 H+ + HCO-3
H2CO3anhydrase
ion H+ and bicarbonate ion
Increased PH and Oxygen-Hemoglobin
Dissociation Curve (bohr effect)

Ion H+

•Increase the stability of hemoglobin (T form)

•Decrease the affinity of hemoglobin to oxygen

•Shift the Dissociation Curve to right

 Bohr effect – the effect of [PH] on
haemoglobin
100%

Higher [CO2] e.g. tissue cells

► curve shift to the right
% saturation of
haemoglobin ► always at a value of lower %
saturation of haemoglobin than the
curve at the left

► haemoglobin has a lower affinity

to O2

partial pressure
of O2 (mmHg)
∴ in actively respiring tissue cells, O2 is more easily released by haemoglobin !
2,3 bisphosphoglycerate
Response of 2,3BPG to chronic anemia and
hypoxia
 Shift to right = low affinity to oxygen
100%

Increased CO2
% saturation of decreased PH
haemoglobin Increased 2.3 BPG
50%

partial pressure
of O2 (mmHg)
26
mmHg
 Shift to left= high affinity to oxygen
100%

decresed CO2
% saturation of increased PH
haemoglobin decresed 2.3 BPG
50%

partial pressure
of O2 (mmHg)
26
mmHg
Hemoglobin and Myoglobin
 An Erythrocyte (RBC)

Normal Values
RBCs, male 4.7-6.1 x 106/µL Practical Values
female 4.2-5.4 x 106/µL 65% of Fe in Hb
Hb, male 13.0-16.0 g/dL 1 g Hb = 3.46 mg Fe
female 12.0-15.0 g/dL 1 mL blood at 15 g/dL Hb = 0.5 mg Fe
Hct, male 42-53% RBC x 3 = Hb
female 37-47% Hb x 3 = Hct
MCH 29±2 pg Microcytic < 81 fL
MCV 81-94 fL Macrocytic > 94 fL
MCHC32-37.5%
Hemoglobin A1c

Diabetes
Increased glucose concentration
Nonenzymatic glycosylation
Associated with diabetic complication
Hemoglobin Genes and Gene Products
Genetics.
In the first 8 weeks of embryonic life the predominant
forms of hemoglobin are:
Hb Gower 1 (ζ2ε2).
Hb Gower 2 (α2ε2).
Hb Portland 1 (ζ2γ2).
By the 12th week embryonic hemoglobin is replaced by
Hb F (α2γ2) which represents 70 – 100% of
hemoglobin in fetal life.
Genetics (2).
Adult hemoglobin Hb A (α2β2) detectable from
16/40, replaces Hb F as predominant hemoglobin
by 6/12 after birth, up to 30% of Hb in fetal life.
In normal adults 96 – 98% of hemoglobin is HbA,
HbF (<1%) constitute a minor component of the
total hemoglobin.
Fetal hemoglobin

Is the main oxygen transport protein in the

fetus during the last seven months of
development in the uterus and in the newborn
until roughly 6 months old.
Baby takes about 2 years to completely
switch over to adult haemoglobin
Function

 Fetal hemoglobin differs most from adult

hemoglobin in that it is able to bind oxygen
with greater affinity than the adult form,
giving the developing fetus better access to
oxygen from the mother's blood stream.
The transfer of oxygen is from the mother
(less tightly bond) to the baby (more tightly
bond).
Characteristic

1. Hemoglobin F is made up of 2 alpha chains and 2

gamma chains
2. Hemoglobin F does not turn into hemoglobin A.
3. Hb F and Hb A are completely different hemoglobin
4. Newborn babies with sickle cell disease make
hemoglobin F and hemoglobin S
 Sickle cell anemia
• Hemoglobin molecules in each red
blood cell carry oxygen from the lungs
to body organs and tissues and bring
carbon dioxide back to the lungs.

• In sickle cell anemia, the hemoglobin

is defective. After hemoglobin
molecules give up their oxygen, some
may cluster together and form long,
rod-like structures. These structures
cause red blood cells to become stiff
and assume a sickle shape.
 Characters of Sickled Red Cell
1. Changing the shape of the RBC from a round disc to
a characteristic crescent (sickle) shape.
2. Sickled red cells cannot squeeze through small blood
vessels.
3. They stack up and cause blockages that deprive
organs and tissues of oxygen-carrying blood.
4. This process produces periodic episodes of pain and
ultimately can damage tissues and vital organs and
lead to other serious medical problems.
Characters of Sickled Red Cell
5. Normal red blood cells live about 120 days in the
bloodstream, but sickled red cells die after about 10 to
20 days.
6. Because they cannot be replaced fast enough, the
blood is chronically short of red blood cells, a
condition called anemia.
 Inheritance
Sickle cell anemia is an autosomal recessive
genetic disorder caused by a defect in the HBB
gene, which codes for hemoglobin.
The presence of two defective genes (SS) is
needed for sickle cell anemia.
 Hemoglobin S differs from normal adult
hemoglobin (hemoglobin A) only by a single
amino acid substitution (a valine replacing a
glutamine in the 6th position of the beta chain of
globing).
When a person has two copies of the S gene
(homozygous SS), he has sickle cell anemia.
 Inheritance
In sickle cell disease, as much as 80% to 100% of the
hemoglobin may be HbS.
A person with one altered S gene will have sickle cell
trait.
In those who have sickle cell trait, 20% to 40% of the
hemoglobin is HbS.
The person does not generally have any symptoms or
health problems but can pass the gene on to his children.
Inheritance
If each parent carries one sickle hemoglobin gene (S) and
one normal gene (A), each child has a 25% chance of
inheriting two defective genes and having sickle cell anemia.

25% chance of inheriting two normal genes and not having

the disease.
50% chance of being an unaffected carrier like the parents.
Pathophysiology

 Normal hemoglobin exists as

solitary units whether oxygenated
or deoxygenated (upper panel).

 In contrast, sickle hemoglobin

molecules adhere when they are
deoxygenated, forming sickle
hemoglobin polymers (lower
panel).
Hemoglobin C
This results when
Hemoglobi
one of the beta nC

subunits is replaced
with beta C
Alpha Alpha Beta S Beta C

The mutation that causes this

change in the beta happens
because a glutamic acid residue
replaces a lysine residue at the sixth
position of the beta globin chain.
Hemoglobin Electrophoresis

Start
position

- +
Hb
A

HbS

Hb
- C
+
cathode migration anode
Hemoglobin Electrophoresis
Beta Thalassemia

β thal major is homozygosity

or compound heterozygosity
resulting in severe phenotype.
β thal minor or trait is
heterozygosity with
asymptomatic phenotype.
Alpha Thalassemia

Father
α α

Mother
α α